Combined gestational age and serum fucose for early prediction of risk for bronchopulmonary dysplasia in premature infants

Objective As the predominant complication in preterm infants, Bronchopulmonary Dysplasia (BPD) necessitates accurate identification of infants at risk and expedited therapeutic interventions for an improved prognosis. This study evaluates the potential of Monosaccharide Composite (MC) enriched with environmental information from circulating glycans as a diagnostic biomarker for early-onset BPD, and, concurrently, appraises BPD risk in premature neonates. Materials and methods The study incorporated 234 neonates of ≤32 weeks gestational age. Clinical data and serum samples, collected one week post-birth, were meticulously assessed. The quantification of serum-free monosaccharides and their degraded counterparts was accomplished via High-performance Liquid Chromatography (HPLC). Logistic regression analysis facilitated the construction of models for early BPD diagnosis. The diagnostic potential of various monosaccharides for BPD was determined using Receiver Operating Characteristic (ROC) curves, integrating clinical data for enhanced diagnostic precision, and evaluated by the Area Under the Curve (AUC). Results Among the 234 neonates deemed eligible, BPD development was noted in 68 (29.06%), with 70.59% mild (48/68) and 29.41% moderate-severe (20/68) cases. Multivariate analysis delineated several significant risk factors for BPD, including gestational age, birth weight, duration of both invasive mechanical and non-invasive ventilation, Patent Ductus Arteriosus (PDA), pregnancy-induced hypertension, and concentrations of two free monosaccharides (Glc-F and Man-F) and five degraded monosaccharides (Fuc-D, GalN-D, Glc-D, and Man-D). Notably, the concentrations of Glc-D and Fuc-D in the moderate-to-severe BPD group were significantly diminished relative to the mild BPD group. A potent predictive capability for BPD development was exhibited by the conjunction of gestational age and Fuc-D, with an AUC of 0.96. Conclusion A predictive model harnessing the power of gestational age and Fuc-D demonstrates promising efficacy in foretelling BPD development with high sensitivity (95.0%) and specificity (94.81%), potentially enabling timely intervention and improved neonatal outcomes.


Introduction
Bronchopulmonary Dysplasia (BPD) is a chronic pulmonary disorder prevalent in very low birth weight infants (VLBWIs; 1000 g ≤ birth weight<1500 g) and extremely low birth weight infants (ELBWIs; birth weight<1000 g).This condition, instigated by impaired lung development and injurious pulmonary responses in preterm infants, imposes severe consequences such as long-term ventilatory reliance, a high mortality rate, and increased susceptibility to lower respiratory infections, airway hyper-reactivity, and growth retardation, thereby drastically undermining their quality of life.With advancements in perinatal medicine improving the survival rates of VLBWIs and ELBWIs, there is a consequent increase in BPD incidence correlating with decreasing birth age and birth weight [1,2].
The multifaceted etiology and pathogenesis of BPD encompass various pathogenic contributors that instigate lung injury and abnormal reparative responses.The diagnosis, as per existing criteria, can only be established at the corrected age of 36 weeks, and the lack of specific treatment modalities underscores the imperative need for sensitive early biomarkers for BPD [3].Metabolomics assays have evidenced altered glucose, lipid, and amino acid metabolism in preterm infants who develop BPD, suggesting that dysregulated glucose metabolism may serve as a predictive marker for BPD onset in preterm neonates [4,5].
Existing reports underscore the pivotal role of glycans in lung development.Animal studies demonstrate that glycan deficiency culminates in pulmonary tissue degradation and impaired lung development [6].Moreover, the absence of fucose inhibits the generation of secretory cells indispensable for airway development [7,8].The intimate relationship between glycans and lung disease pathogenesis has been well-documented [9,10].Glycan degradation is implicated in pulmonary injury and influences its prognosis.In acute lung injury, the cellular glycan structures can be shed into the bloodstream, hence, any aberration in blood glycan composition can serve as a marker of pathology.Therefore, variations in monosaccharide content in premature infants may offer an efficient approach for early BPD diagnosis in this vulnerable population.
In this study, we analyzed the concentrations of serumfree monosaccharides and degraded monosaccharides in premature infants through High-performance Liquid Chromatography (HPLC), looking for the novel diagnostic biomarker for BPD.Moreover, we integrating clinical data for enhanced diagnostic precision.This study aspired to construct an early diagnostic model to assess the risk and improve the prognosis of Bronchopulmonary Dysplasia (BPD).

Subjects
The criteria for participant inclusion were as follows: (1) Gestational age ≤ 32 weeks; (2) Hospital admission within 1 hour post-birth, with a subsequent hospital stay exceeding 28 days; (3) Complete clinical data.Exclusionary factors included patients with chromosomal abnormalities, inherited metabolic diseases, or congenital developmental malformations.Our study included a cohort of 234 preterm infants.The research spanned from June 2020 to October 2023.4 ml fasting venous blood samples from premature infants were collected when they were 1 week after birth, and centrifuged for 5 min at 13000 r/min.We took 200 μl serum and preserved it bellow at − 80 °C for the following analysis.At the same time, we collected detailed clinical information of all subjects occurred during hospitalization.In accordance with the Bronchopulmonary Dysplasia definition and grading criteria revised by the National Institute of Child Health and Human Development (NICHD) in 2018, these patients were classified into a non-BPD group (n = 166) and a BPD group (n = 68), with 48 designated as mild BPD (Grade I), and 20 as moderate to severe BPD (Grades II, III or IIIA) [3].
The study received approval from the Medical Ethics Committee of Qingdao University's Affiliated Hospital (QYFY WZLL 28446), with informed consent obtained from all participating guardians.

Serum monosaccharide degradation determination
For each serum sample, 5 μL was combined with Rha (1 mg/mL), 10 μL of HCl (6 mol/L), and following mixing, it underwent PCR degradation (100 °C for 10 min).Subsequent addition of (1/6.3)NaOH solution and 0.5 mol/L PMP solution occurred before carrying out PCR derivatization and purification as described in 2.2.A 50 μL aliquot of the supernatant was used for HPLC analysis.This method, known as sugar fingerprinting technology, is a patented technique of our laboratory [11].
Rha was utilized as the internal standard to maintain the consistency of experimental operations.The monosaccharide standard mixture underwent HPLC analysis, producing the HPLC chromatogram depicted in Fig. 1A.Using the peak areas of various monosaccharide standard concentration gradients, the standard curve and regression equation were established.HPLC was used for the analysis of serum-free monosaccharides and degraded monosaccharides, obtaining the corresponding HPLC chromatograms (Fig. 1B and C).These data allowed for a precise calculation of each monosaccharide or degraded monosaccharide concentration for further analysis.

Statistical methods
Data was processed using SPSS 25.0, and figures were generated with GraphPad Prism 8. Continuous variables adhering to the normal distribution were analyzed using the t-test and expressed as mean ± standard deviation (x ± s).Non-normal variables were subjected to the Mann-Whitney U test and presented as median and interquartile range [M (Q1, Q3)].Qualitative data, reported as the case number and percentage, were assessed using the chi-square or Fisher's exact test.The diagnostic potential of distinct monosaccharides for BPD was examined via ROC curves, with diagnostic efficacy evaluated using the AUC.A p-value less than 0.05 denotes statistical significance.

Clinical data comparison between BPD and non-BPD groups
The study comprised 234 subjects, 166 in the non-BPD group and 68 in the BPD group, yielding a BPD incidence of 29.06%.Mild BPD accounted for 70.59% (48/68) within the BPD group, while moderate or severe BPD made up 29.41% (20/68).
Clinical data comparisons revealed lower gestational age (P = 0.000) and birth weight (P = 0.000) in the BPD group than in the non-BPD group.Both invasive and non-invasive ventilation durations were longer in the BPD group (P = 0.000).The BPD group exhibited a higher prevalence of Ductus Arteriosus (PDA) and gestational hypertension (P = 0.01).No statistically significant differences in sex, caesarean section, prelabor rupture, gestational diabetes mellitus, Small for Gestation (SGA), septicemia, Extrauterine Growth Restriction (EUGR), Pulmonary Surfactant (PS), and Respiratory Distress Syndrome (RDS) were found (P > 0.05) (Table 1).

Analysis of the diagnostic efficacy of free monosaccharides and degraded monosaccharides in serum
We examined the concentration disparity of free and degraded monosaccharides in serum between the BPD and non-BPD groups.The BPD group exhibited significantly higher concentrations of Glc-F (P = 0.000), Man-F(P = 0.000), Man-D(P = 0.000), GalN-D(P = 0.001), Glc-D(P = 0.000), Gal-D(P = 0.015), and Fuc-D (P = 0.000).No statistically significant differences were observed in G/M and GlcN-D concentrations (P > 0.05) (Table 2).

Development of a BPD prediction model in premature infants
Considering the disparities in clinical data and monosaccharide concentrations between the BPD and non-BPD groups, a predictive model Z = 123.975-4.325*gestationalage + 0.024*Fuc-D was devised via logistic regression analysis.In comparison to individual factor predictive models, the amalgamation of gestational age and Fuc-D significantly bolstered the diagnostic efficiency, yielding an AUC of 0.96, sensitivity of 95.0%, and a specificity of 94.81%.Consequently, the predictive model, contingent on gestational age and Fuc-D offered substantial diagnostic value for BPD in premature infants (Fig. 3).

Comparative analysis of serum free and degraded monosaccharides in mild BPD vs. moderate to severe BPD
Statistical examination revealed no significant differences in Glc-F, Man-F, G/M, Man-D, GlcN-D, GalN-D and Gal-D levels between the mild BPD group and the moderate-to-severe BPD group (P > 0.05).Nevertheless, the concentrations of Glc-D (P = 0.000) and Fuc-D (P = 0.005) were lower in the moderate-to-severe BPD group compared to the mild BPD group, a difference that proved statistically significant.(Table 3).

Discussion
Bronchopulmonary Dysplasia (BPD) arises due to an amalgamation of adverse influences during lung development, such as infection, volume injury, barotrauma, hyperoxia injury, and Patent Ductus Arteriosus (PDA), alongside genetic predispositions [12].With intrinsically immature lung development, premature infants inherently bear a higher susceptibility to BPD.Our study identified a BPD frequency of 29.06%.However, the global incidence of BPD among extremely premature infants from 2006 to 2017 oscillated between 10 and 89%, a variability likely attributed to discrepancies in gestational age, birth weight, clinical diagnostic parameters, and preterm infant management strategies [13].
The comparative analysis of clinical data between BPD and non-BPD groups uncovered statistically significant differences in gestational age, birth weight, duration of invasive and non-invasive mechanical ventilation, PDA, and gestational hypertension.Predominantly, BPD occurs in very low birth weight infants (VLBWI) and extremely low birth weight infants (ELBWI) with gestational ages less than 32 weeks.The incidence rate exhibited a negative correlation with gestational age and birth weight (Table 1) [14].Premature infants' lungs, typically at tubular or vesicle stages of development at birth, exhibit deficient synthesis and secretion of pulmonary surfactant, diminished lung compliance, and weak antioxidant capabilities, rendering them prone to endogenous and exogenous damage [15].BPD ensues when alveolar and microvascular development processes are impeded [16].Mechanical ventilation, especially in relation to volume pressure injury, has been recognized as a primary contributor to lung injury, thereby an independent risk factor for BPD [17,18].The connection between BPD and preeclampsia, however, remains somewhat enigmatic.Previous reports have suggested that preeclampsia can  result in placental dysfunction and an imbalance between pro-and anti-angiogenic factors [19], potentially impacting fetal alveolar and lung capillary development and subsequently increasing BPD risk [20,21].Our findings support this hypothesis, as the incidence of maternal hypertension during pregnancy was higher in the BPD group.Moreover, the incidence of PDA was greater in the BPD group.Given the hypoplastic state of the smooth muscle and the lack of intimal cushion, an immature ductus arteriosus might not close timely after birth, causing enhanced blood flow to the lungs, pulmonary edema, lung congestion, and subsequent exacerbation of pulmonary inflammation, declining lung function, and reduced gas exchange.Although the precise role of PDA in BPD onset and progression remains elusive, the risk of BPD in premature infants with PDA was found to be 6.266 times that of non-PDA counterparts, with the BPD risk escalating alongside PDA shunt volume and duration [22,23].Alveolar epithelial cells and capillary endothelial cells exhibit a dense glycan structure.Under physiological conditions, this glycan structure dynamically balances synthesis and degradation, offering functions such as anti-inflammation, vascular permeability maintenance, and endothelial function protection [24].The levels of free and degraded monosaccharides in serum mirror alterations in the structure and quantity of these glycans.It has been reported that the degradation of alveolar epithelial glycans predominantly occurs in patients with acute lung injury, causing pulmonary surfactant (PS) dysfunction and correlating with the duration of mechanical ventilation [25].Disturbances in glycan structure and the inflammatory response form two pivotal facets of acute lung injury [26,27].Several studies have emphasized that concentrations of glycan degradation products rise in the peripheral blood of acute lung injury patients [28].In the event of acute lung injury, glycan degradation can promote and augment the deformation and adhesion abilities of circulating inflammatory cells, facilitating their migration to the pulmonary interstitium and alveoli [29].Furthermore, glycan degradation prompts the production of numerous inflammatory mediators, such as oxygen free radicals, lipids, and peptides.These mediators inflict direct damage upon alveolar epithelial cells, stromal cells, and capillary basement membranes, enhancing vascular permeability [30], impeding pulmonary blood vessel development, inciting PS inactivation, and obstructing alveolarization [31].These inflammatory mediators also display extensive biological activity, possibly inciting their re-release [32].The continued presence of proinflammatory factors and chemokines triggers an unregulated 'waterfall' secondary inflammatory cascade [33], leading to accelerated lung glycan degradation [34], hindered repair and reconstruction function, subsequent pulmonary vascularization disorders, and abnormal repair, ultimately culminating in BPD.In this study, the BPD group demonstrated higher concentrations of Man-D, GalN-D, Glc-D, Gal-D, Fuc-D, Man-F, and Glc-F than the non-BPD group, suggesting that monosaccharide levels in premature infants could reflect lung injury and abnormal repair processes.Our research confirmed that monosaccharide content might serve as a potent early diagnostic indicator for BPD in premature infants, with the identified monosaccharides (Man-D, GalN-D, Glc-D, Gal-D, Fuc-D, Man-F, and Glc-F) demonstrating substantial predictive value for BPD.
This study has formulated a prediction model for BPD in premature infants, utilizing logistic regression analysis based on gestational age and Fuc-D content.As a glycan structure modification, Fuc contributes unique functional properties to sugar chains and plays a role in lung development and cell differentiation regulation.Deficiency of Fuc in lungs has been associated with pulmonary dysplasia [35], and it has also been implicated in the scavenging of oxygen free radicals, exhibiting antioxidant and anti-inflammatory effects.Previous studies have shown lower levels of Fuc-D in the urine metabolites of BPD group preterm infants compared to non-BPD counterparts [36].Our investigation noted that the concentrations of Glc-D (P = 0.000) and Fuc-D (P = 0.005) were lower in the moderate-to-severe BPD group than in the mild BPD group.It was postulated that severe BPD premature infants, with heightened oxidative stress, consume more Glc and Fuc due to the presence of a large quantity of reactive oxygen radicals, leading to severe oxidative stress responses and further damage to immature lungs [37].Nevertheless, the precise association between serum free and degraded monosaccharide levels and varying degrees of BPD warrants additional study.
In the domain of BPD prediction models, frequent predictors include birth weight, gestational age, sex, 5-minute Apgar score, respiratory distress syndrome, mechanical ventilation, antenatal steroids, maternal hypertensive disorders, surfactant, and patent ductus arteriosus [38].Emerging markers for BPD have been reported to encompass lung ultrasound and urinary markers [39,40].Approximately 30% of related studies construct prediction models via univariable analysis, potentially overlooking crucial predictors.Consequently, multi-factorial prediction models are gaining wider exploration.Cai et al. identified 10 independent risk factors to construct their model, achieving an AUC value of 0.965 (sensitivity: 93.7%; specifity: 91.3%) [23].Jassem-Bobowicz et al. established a model based on four factors, obtaining an AUC of 0.932 [3], while Shim et al. achieved high predictability (90.8%) using clinical parameters collected within 1 hour of birth [40].In our study, the developed prediction model, based on the combination of gestational age and Fucose, delivered an AUC of 0.96, a high sensitivity of 95.0%, and a specificity of 94.81%.
In conclusion, the link between monosaccharides and BPD was investigated for the first time, suggesting Fucose as a novel marker for BPD in premature infants.Despite promising results, the main limitation of this study lies in the small sample size.Therefore, external validation through multi-center prospective studies is indispensable to further assess the generalizability of this prediction model.

Fig. 1 A
Fig. 1 A HPLC chromatogram of monosaccharide standard; B HPLC chromatogram of serum free monosaccharides.C HPLC chromatogram of serum degraded monosaccharides

Fig. 3
Fig. 3 Efficacy evaluation of the BPD prediction model

Table 1
Clinical data comparison between BPD and Non-BPD GroupsSGA small for gestational age, PDA ductus arteriosus, EUGR extrauterine growth restriction, PS pulmonary surfactant, RDS respiratory distress syndrome, M ± SD mean ± standard deviation, IQR interquartile range

Table 2
Comparison of monosaccharide content in the BPD and non-BPD groups Glc-F free glucose, Man-F free mannose, G/M free glucose/ free mannose, Man-D degraded mannose, GlcN-D degraded glucosamine, GalN-D degraded galactosamine, Glc-D degraded glucose, Gal-D degraded galactose, Fuc-D degraded fucose, M ± SD mean ± standard deviation, IQR interquartile range Fig. 2 ROC curve of serum free and degraded monosaccharides